This project was partially funded by USDA Multi-state project W4112 and UC Davis Jastro Shields award to SM.
The authors would like to extend their appreciation to Central Valley Meat, Inc. for providing the bovine ovaries used in all experiments. The authors also thank Olivia Silvera for assistance with ovary processing and follicle isolation.

Bovine (Bos taurus) ovaries were sourced from a local abattoir and transported to the laboratory within 6 h of collection. Due to the large number of animals processed in the facility, the age, breed, and stage of the estrus cycle of the animals are unknown. Because no live animals were used in these experiments, an approved animal care and use protocol was not required.
1. Preparation of equipment and reagents
<ol>
	<li>Cover a 2 ft wide section of a lab bench with...

The authors have nothing to disclose.

The present protocol details a reproducible method to retrieve early stage preantral follicles, specifically at primary and early secondary stages, from the bovine ovary. This protocol builds on previous reports20,25,30,34,35,36 and provides optimizations that result in the isolation of a meaningful number of follicles from a...

Ovarian follicles are the functional units of the ovary, responsible for production of the gamete (oocyte) as well as hormones critical for reproductive function and overall health. Primordial follicles form in the ovary during fetal development or in the neonatal period depending on the species1, and they constitute a female&#39;s ovarian reserve. Follicular growth begins with the activation of primordial follicles that leave the resting pool and enter the growing phase. Preantral folliculogenesis, encompassing all follicle stages before antrum development, is a highly dynamic process that requires synchronous morphological and metabolic changes in the oocyte and the surrounding granulosa cells, driven by tight communication between these two cell types2,3. Preantral follicles constitute the majority of the follicular units found in the ovary at any given time4. Development through the preantral stages of folliculogenesis is estimated to be several weeks longer than antral development5,6, and this time is necessary for the oocyte and somatic cells to acquire sufficient maturity to enter the final stage of development (i.e., the antral stage), and prepare for ovulation, fertilization, and embryonic development7,8,9.
Much of the current knowledge about ovarian preantral folliculogenesis comes from mouse models10,11,12,13, due in part to the ease in recovering a large number of these follicles from a smaller and less fibrous ovary. Although reports of isolation of large numbers of preantral follicles from bovine ovaries date back approximately 30 years14, a more complete understanding about the processes regulating the development of these early-stage follicles has remained unrealized, largely due to the lack of optimized, efficient, and repeatable methods to retrieve sufficient numbers of viable preantral follicles, particularly at early stages of development. With the increasing interest in preserving the ovarian reserve for future use in assisted reproduction in humans, cows become an attractive model due to their more similar ovarian structure15. However, the bovine ovary is markedly richer in collagen compared to the mouse ovary16, making mechanical isolation using methods described for the mouse very inefficient. Efforts to expand fertility preservation techniques include complete in vitro growth of preantral follicles to the antral stage, followed by in vitro maturation (IVM) of the enclosed oocytes, in vitro fertilization (IVF), and embryo production and transfer17. Thus far, this entire process has only been achieved in mice18. In cattle, the progress toward follicle growth in vitro is limited to a few reports with variable follicle stages at the start of culture, as well as variable length of culture between protocols17,19.
The methods described in the literature for the harvest of preantral follicles from the bovine ovary have mostly used mechanical and enzymatic techniques, either isolated or in combination2,14,17,20. The first report of a protocol for bovine preantral follicle isolation used a tissue homogenizer and serial filtration to process whole ovaries20. This study was followed by reports combining mechanical and enzymatic procedures that utilized collagenase14. A recurrent theme when utilizing collagenase to digest the ovarian tissue is the potential risk for damage of the follicular basement membrane, which may compromise follicle viability14,21,22,23. Therefore, different combinations of mechanical methods have been employed, such as the use of a tissue chopper and repeated pipetting or a tissue chopper combined with homogenization20,24,25,26. Another mechanical technique that has been described utilizes needles to dissect preantral follicles directly from the ovarian tissue, which is especially useful for isolating larger (&#62;200 &#181;m) secondary follicles. However, this process is time-consuming, inefficient for isolating smaller preantral follicles, and is skillset-dependent when attempted in bovine ovaries19,27,28.
Taking advantage of the different techniques described in the literature, this protocol aimed to optimize the isolation of preantral follicles from single bovine ovaries in a simple, consistent, and efficient manner that avoids incubation in enzymatic solutions. Improving the methods to isolate preantral follicles will provide an opportunity to enhance the understanding of this stage of folliculogenesis and enable the development of effective culture systems to develop preantral follicles to the antral stage. The detailed procedures described herein for the isolation of preantral follicles from a large mammal such as the bovine species will be vital for researchers aiming to study early folliculogenesis in a non-murine species that is translatable to humans.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-3/4&#34; Soda Lime Disposable Glass Pasteur Pipette</td>
			<td>Duran Wheaton Kimble</td>
			<td>63A54</td>
			<td>Pasteur pipette that can be used to dislodge follicles from debris while searching within the petri dish</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Diluted to 4%; fixation of follicles for immunostaining</td>
		</tr>
		<tr>
			<td>20 mL Luer-lock Syringe</td>
			<td>Fisher Scientific</td>
			<td>Z116882-100EA</td>
			<td>Syringe used with the 18 G needle to dislodge follicles from the 40 &#956;m cell strainer</td>
		</tr>
		<tr>
			<td>#21 Sterile Scalpel Blade</td>
			<td>Fisher Scientific</td>
			<td>50-365-023</td>
			<td>Used to cut the ovaries and remove the medula</td>
		</tr>
		<tr>
			<td>40 &#956;m Cell Strainer</td>
			<td>Fisher Scientific&#160;</td>
			<td>22-363-547</td>
			<td>Used to filter the filtrate from the 300 &#956;m cell strainer</td>
		</tr>
		<tr>
			<td>104 mm Plastic Funnel</td>
			<td>Fisher Scientific</td>
			<td>10-348C</td>
			<td>Size can vary, but ensure the cheese cloth is cut appropriately and that the ovarian homogenate will not spill over</td>
		</tr>
		<tr>
			<td>300 &#956;m Cell Strainer</td>
			<td>pluriSelect&#160;</td>
			<td>43-50300-03</td>
			<td>Used to filter the filtrate from the cheese cloth&#160;</td>
		</tr>
		<tr>
			<td>500 mL Erlenmeyer Flask</td>
			<td>Fisher Scientific</td>
			<td>FB500500</td>
			<td>Funnel and flask used to catch filtrate from the cheese cloth&#160;</td>
		</tr>
		<tr>
			<td>Air-Tite Sterile Needles 18 G</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-817-151</td>
			<td>18 G offers enough pressure to dislodge follicles from the 40 &#956;m cell strainer</td>
		</tr>
		<tr>
			<td>Air-Tite Sterile Needles 27 G 13 mm</td>
			<td>Fisher Scientific</td>
			<td>14-817-171</td>
			<td>Needles that can be used to manipulate any debris in which follicles are stuck</td>
		</tr>
		<tr>
			<td>BD Hoechst 33342 Solution</td>
			<td>Fisher Scientific</td>
			<td>BDB561908</td>
			<td>Fluorescent DNA stain</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A7030-100G&#160;</td>
			<td>Component of follicle wash media</td>
		</tr>
		<tr>
			<td>Cheese Cloth</td>
			<td>Electron Microscopy Sciences</td>
			<td>71748-00</td>
			<td>First filtering step of the ovarian homogenate meant to remove large tissue debris</td>
		</tr>
		<tr>
			<td>Classic Double Edge Safety Razor Blades</td>
			<td>Wilkinson Sword</td>
			<td>N/A</td>
			<td>Razor blades that fit the best in the McIlwain Tissue Chopper and do not dull quickly</td>
		</tr>
		<tr>
			<td>Donkey-Anti-Rabbit Secondary Antibody, Alexa Fluor 488</td>
			<td>Fisher Scientific</td>
			<td>A-21206</td>
			<td>Secondary antibody for immunostaining</td>
		</tr>
		<tr>
			<td>Eisco Latex Pipette Bulbs</td>
			<td>Fisher Scientific</td>
			<td>S29388</td>
			<td>Rubber bulb to use with Pasteur pipettes</td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Component of follicle wash media</td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>VWR</td>
			<td>10032-336</td>
			<td>Homogenize the ovarian tissue to release follicles&#160;</td>
		</tr>
		<tr>
			<td>ImageJ/Fiji</td>
			<td>NIH</td>
			<td>v2.3.1</td>
			<td>Software used for analysis of fluorescence-immunolocalization</td>
		</tr>
		<tr>
			<td>McIlwain Tissue Chopper</td>
			<td>Ted Pella</td>
			<td>10184</td>
			<td>Used to cut ovarian tissue small enough for homogenization</td>
		</tr>
		<tr>
			<td>Microscope - Stereoscope</td>
			<td>Olympus</td>
			<td>SZX2-ILLT</td>
			<td>Dissection microscope used for searching and harvesting follicles from the filtrate</td>
		</tr>
		<tr>
			<td>Microscope - Inverted</td>
			<td>Nikon</td>
			<td>Diaphot 300</td>
			<td>Inverted microscope used for high magnification brightfield visualization of isolated follicles</td>
		</tr>
		<tr>
			<td>Microscope - Inverted</td>
			<td>ECHO</td>
			<td>Revolve R4</td>
			<td>Inverted microscope used for high magnification brightfield and epifluorescence visualization of isolated follicles</td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma-Aldrich</td>
			<td>M8410-1L</td>
			<td>Oil to cover the drops of follicle wash medium to prevent evaporation during searching</td>
		</tr>
		<tr>
			<td>Non-essential Amino Acids (NEAA)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td>Component of follicle wash medium</td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-001</td>
			<td>Reagent for immunostaining blocking buffer</td>
		</tr>
		<tr>
			<td>Nunc 4-well Dishes for IVF</td>
			<td>Thermo Fisher Scientific</td>
			<td>144444</td>
			<td>4-well dishes for follicle isolation and washing</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution 100x</td>
			<td>Gibco</td>
			<td>15-140-122</td>
			<td>Component of follicle wash medium</td>
		</tr>
		<tr>
			<td>Petri Dish 60 mm OD x 13.7 mm</td>
			<td>Ted Pella</td>
			<td>10184-04</td>
			<td>Petri dish that fits the best in the McIlwain Tissue Chopper</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP665-1</td>
			<td>Washing buffer for ovaries and follicles</td>
		</tr>
		<tr>
			<td>Plastic Cutting Board</td>
			<td>Fisher Scientific</td>
			<td>09-002-24A</td>
			<td>Cutting board of sufficient size to safely cut ovaries</td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone (PVP)</td>
			<td>Fisher Scientific</td>
			<td>BP431-100</td>
			<td>Addition of PVP (0.1% w/v) to PBS prevents follicles from sticking to the plate or each other&#160;</td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36930</td>
			<td>Mounting medium for fluorescently labeled cells or tissue</td>
		</tr>
		<tr>
			<td>Qiagen RNeasy Micro Kit</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td>RNA column clean-up kit</td>
		</tr>
		<tr>
			<td>R</td>
			<td>The R Foundation</td>
			<td>v4.1.2</td>
			<td>Statistical analysis software</td>
		</tr>
		<tr>
			<td>Rabbit-Anti-Human Cx37/GJA4 Polyclonal Antibody</td>
			<td>Abcam</td>
			<td>ab181701</td>
			<td>Cx37 primary antibody for immunostaining</td>
		</tr>
		<tr>
			<td>RevertAid RT Reverse Transcription Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>K1691</td>
			<td>cDNA synthesis kit</td>
		</tr>
		<tr>
			<td>Rstudio</td>
			<td>RStudio, PBC</td>
			<td>v2021.09.2</td>
			<td>Statistical analysis software</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide Solution (1N/Certified)</td>
			<td>Fisher Scientific</td>
			<td>SS266-1</td>
			<td>Used to increase media pH to 7.6-7.8</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (NaPyr)</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td>Component of follicle wash medium</td>
		</tr>
		<tr>
			<td>Square Petri Dish 100 mm x 15 mm&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>60872-310</td>
			<td>Gridded petri dishes allow for more efficient identification of follicles&#160;</td>
		</tr>
		<tr>
			<td>SsoAdvanced Universal SYBR Green Supermix</td>
			<td>BioRad</td>
			<td>1725271</td>
			<td>Mastermix for PCR reaction</td>
		</tr>
		<tr>
			<td>Steritop Threaded Bottle Top Filter</td>
			<td>Sigma-Aldrich</td>
			<td>S2GPT02RE</td>
			<td>Used to sterilize follicle wash medium</td>
		</tr>
		<tr>
			<td>SYBR-safe DNA gel stain</td>
			<td>Thermo Fisher Scientific</td>
			<td>S33102</td>
			<td>Staining to visual PCR products on agarose gel</td>
		</tr>
		<tr>
			<td>TCM199 with Hank&#8217;s Salts</td>
			<td>Gibco</td>
			<td>12-350-039</td>
			<td>Component of follicle wash medium</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-100</td>
			<td>Detergent for immunostaining permeabilization buffer</td>
		</tr>
		<tr>
			<td>Trizol reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td>RNA isolation reagent</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Gibco</td>
			<td>15-250-061</td>
			<td>Used for testing viability of isolated follicles</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td></td>
			<td></td>
			<td>Detergent for immunostaining wash buffer</td>
		</tr>
		<tr>
			<td>Warmer Plate Universal</td>
			<td>WTA</td>
			<td>20931</td>
			<td>Warm plate to keep follicles at 38.5 &#176;C while searching under the microscope</td>
		</tr>
		<tr>
			<td>Wiretrol II Calibrated Micropipets</td>
			<td>Drummond</td>
			<td>50002-005</td>
			<td>Glass micropipettes to manipulate follicles</td>
		</tr>
	</tbody>
</table>

isolation of small preantral follicles from the bovine ovary using a combination of fragmentation, homogenization, and serial filtration

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and endangered species. Research has been mostly limited to antral and large preantral follicles due to difficulty in the isolation of smaller preantral follicles, especially in large mammals such as bovine species. This work presents an efficient approach to retrieve large numbers of small preantral follicles from a single bovine ovary. The cortex of individual bovine ovaries was sliced into 500 &#181;m cubes using a tissue chopper and homogenized for 6 min at 9,000-11,000 rpm using a 10 mm probe. Large debris was separated from the homogenate using a cheese cloth, followed by serial filtration through 300 &#181;m and 40 &#181;m cell strainers. The contents retained in the 40 &#181;m strainer were rinsed into a search dish, where follicles were identified and collected into a drop of medium. The viability of the collected follicles was tested via trypan blue staining. This method enables the isolation of a large number of viable small preantral follicles from a single bovine ovary in approximately 90 min. Importantly, this method is entirely mechanical and avoids the use of enzymes to dissociate the tissue, which may damage the follicles. The follicles obtained using this protocol can be used for downstream applications such as isolation of RNA for RT-qPCR, immunolocalization of specific proteins, and in vitro culture.

Overview and critical steps 
Using this protocol, small bovine preantral follicles can be reliably isolated from single ovaries in experimentally relevant numbers. From a total of 30 replicates, an average of 41 follicles were obtained per replicate, with a range of 11 to 135 follicles (Figure 4A). In 14 replicates, the follicles were characterized for stage of development as previously described26 by measuring the follicle diameter using a 1 &#1...

Watch this Scientific Journal Video about Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration at JoVE.com

Isolation of Small Preantral Follicles from the Bovine Ovary Using a Combination of Fragmentation, Homogenization, and Serial Filtration

Advancing the study of preantral folliculogenesis requires efficient methods of follicle isolation from single ovaries. Presented here is a streamlined, mechanical protocol for follicle isolation from bovine ovaries using a tissue chopper and homogenizer. This method allows collection of a large number of viable preantral follicles from a single ovary.

Understanding the full process of mammalian folliculogenesis is crucial for improving assisted reproductive technologies in livestock, humans, and ...

This protocol allows for the efficient and repeatable isolation of early stage pre-antral follicles from a single bovine ovary and it creates new opportunities for the study of ovarian folliculogenesis in a non-bovine species. By using a greater number of follicles from a single ovary, this technique enables in vivo treatments before ovary harvest, or obtention of multiple samples over time from the same animal via ovarian biopsy. Isolation and cryo-preservation of early stage pre-antral follicles can be a valuable option for fertility preservation in young women undergoing gonadotoxic treatment such as chemotherapy, as well as germplasm preservation from endangered species.Demonstrating the procedure will be Stephanie McDonnell, Amanda Morton, and Juliana Candelaria, PhD students from my laboratory. To begin, transfer ovaries in the laboratory to warm, sterile PBS containing Pen-Strep and select ovaries with many small antral follicles and no prominent corpus lutetium. Remove any excess connective tissue and fat from the ovaries using scissors.Transfer one ovary to the cutting board on the bench paper. Using a scalpel, cut the ovary in half longitudinally from one ligament attachment site to the opposite attachment site. Keep one half of the ovary on the cutting board to be processed and place the other half of the ovary back into warm PBS containing Pen-Strep.To remove the medulla, slice along the curvature of the ovary approximately 2 millimeter away from the surface without cutting through the cortex with the exposed medulla facing upward. Slice down through the cut to remove the majority of the medulla. Flip the ovary half over so that the epithelium faces upward and use the scalpel to finish cutting the medulla away from the cortex and trim away any remaining white connective tissue around the edge of the ovary piece that was connected to the ligaments.Once the majority of the medulla is removed, use the scalpel to cut the cortex to approximately 1-millimeter thickness while manipulating the scalpel with small back-and-forth motions to shave away the remainder of the medulla, then cut each ovary cortex half into two pieces. Keep the cortex pieces in warm PBS containing Pen-Strep until ready to chop. Transfer a single piece of the cortex to the Petri dish on the tissue chopper and wet the tissue with three or four drops of warm PBS containing Pen-Strep.While holding the piece of tissue steady with a pair of forceps, press the Reset button once to start the tissue chopper. After the entire piece of the cortex has been cut into strips, use the blade holder knob to lift the blade off the Petri dish and the forceps to remove any tissue from the blade. After rotating the plate holder by 90 degrees, again, press the Reset button and pass the blade entirely through the tissue strips.Use the blade holder knob to lift the blade off the Petri dish and the forceps to remove any tissue from the blade as demonstrated previously. Using a transfer pipette and warm PBS containing Pen-Strep, wash the chopped tissue into a pre-warmed 50-milliliter conical tube. Return the conical tube to the water or bead bath to keep the chopped tissue warm while repeating the chopping procedure with the other three halves of the ovary.For best cutting results, use the nut driver to remove the nut from the chopping arm and remove the washer and blade clasp. Using forceps, remove the blade from the chopping arm. Flip it over so that the unused edge is facing the Petri dish and place it back onto the chopping arm.After ensuring that the homogenizer unit is plugged in and the speed is set to the second bar, insert the 10-millimeter generator probe into the unit according to the manufacturer's specifications. Next, set a timer for 1 minute and insert the probe into the 50-milliliter conical tube containing the chopped cortex tissue from one ovary and enough PBS containing Pen-Strep to fill the tube to the 25 milliliter line. The depth to which the probe is inserted must be 1/3 of the liquid's height measured from the bottom of the chamber.Once the timer starts, turn on the homogenizer, ensuring that the bottom of the probe does not touch the tube and hold the tube still while the homogenizer is turned on. After 1 minute of homogenization, remove the probe from the tube, then remove any connective tissue clogging the venting holes in the space between the rotor knife and rotor tube or cortex pieces stuck in the probe using forceps and place them back into the tube. Pour the dispersed tissue into the cheesecloth-covered funnel, inserted into the Erlenmeyer flask and rinse the contents of the tube into the funnel using warm PBS.Finally, rinse the cheesecloth with warm PBS. Force the liquid in small fragments to pass through the holes of the cloth by twisting the cheesecloth around the tissue fragments down and squeezing until all excess fluid and tissue are removed from the cheesecloth. After reopening the cheesecloth over the funnel, rinse the cheesecloth with PBS containing Pen-Strep using a transfer pipette, and again, squeeze any residual tissue fragments through the cloth.Using a hemostat, hold the 300-micrometer cell strainer over a 200-milliliter beaker and pour the filtrate in the Erlenmeyer flask through the cell strainer. If the cell strainer becomes clogged with tissue, gently tap it against the beaker, then turn the strainer upside down and tap out the large tissue debris onto the bench paper. Rinse the contents of the flask using warm PBS containing Pen-Strep until no tissue fragments remain and pour it into the cell strainer.Next, while holding the 40-micrometer cell strainer with a hemostat over a second 200-milliliter beaker, pour all the initial filtrate present in the first 200-milliliter beaker through the strainer, then rinse the contents of the beaker using warm PBS containing Pen-Strep until no tissue fragments remain and pour it into the cell strainer. After fitting the 18 gauge needle to the 20-milliliter syringe, fill the syringe with follicle wash medium. With a hemostat, hold the 40-micrometer cell strainer upside down over a square Petri dish and use the syringe to wash out the contents of the cell strainer into the dish.Transfer the square Petri dish to a stereoscope with a warm stage set to 38.5 degrees Celsius with a magnification set between 1.25 and 3.2x. After identifying follicles from the square Petri dish, transfer the follicle to wash medium drops using the micropipette. The total number of isolated pre-antral follicles per replicate using 6-minute tissue homogenization shows an average of 41 follicles per replicate, ranging from 11 to 135 follicles.Assessment of the developmental stage of 476 isolated follicles showed that the majority were in the primary stage of development, measuring 40 to 79 micrometers in containing one layer of cuboidal granulosa cells. Homogenization of the ovarian cortex for 6 minutes yielded a greater number of pre-antral follicles compared to 5 minutes of homogenization at the same speed. Transcript expression of the granulosa cell marker FSH receptor and germ cell marker DAZL in the follicles was evaluated using reverse transcription quantitative PCR demonstrating that both primary and early secondary follicles expressed FSH receptor and DAZL transcripts.Immunofluorescent localization of CX37 in isolated pre-antral follicles at both the primary and early secondary stages shows that CX37 was localized to the O plasm, being absent from the nucleus of the O site and to the membrane of the granulosa cells. In our experience, proper dissection of a thin layer of cortical tissue, chopping the tissue to the right size, and proper homogenization are critical steps to ensure release of viable follicles from the tissue. Isolated pre-antral follicles can be used to answer questions such as regulation of pre-antral follicle growth and the nature of interactions between granulosa cells and the oocyte, thus facilitating the development of more efficient culture conditions.

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JoVE Journal

Biology

3.0K visualizações.  University of California Davis. Este protocolo permite o isolamento eficiente e repetível de folículos pré-antrais em estágio inicial de um único ovário bovino e cria novas oportunidades para o estudo da foliculogênese ovariana em uma espécie não bovina. Ao utilizar um maior número de folículos de um único ovário, esta técnica permite tratamentos in vivo antes da colheita do ovário, ou a obtenção de múltiplas amostras ao longo do tempo do mesmo animal através de biópsia ovariana. O isolamento e a criopre...